The invention concerns a composition, preferably a pharmaceutical composition, of hedgehog proteins and its use.
Hedgehog (hh) proteins are understood as a family of secreted signal proteins which are responsible for the formation of numerous structures in embryogenesis (J. C. Smith, Cell 76 (1994) 193-196, N. Perrimon, Cell 80 (1995) 517-520, C. Chiang et al., Nature 83 (1996) 407, M. J. Bitgood et al., Curr. Biol. 6 (1996) 298-304, A. Vortkamp et al., Science 273 (1996) 613, C. J. Lai et al., Development 121 (1995) 2349). During its biosynthesis a 20 kDa N-terminal domain and a 25 kDa C-terminal domain are obtained after cleavage of the signal sequence and autocatalytic cleavage. In its natural form the N-terminal domain is modified with cholesterol or palmitoyl (J. A. Porter et al., Science 274 (1996) 255-259, Pepinski et al., J. Biol.Chem. 273 (1998) 14037-14045). In higher life-forms the hh family is composed of at least three members namely sonic, indian and desert hh (shh, ihh, dhh; M. Fietz et al., Development (Suppl.) (1994) 43-51). Differences in the activity of hedgehog proteins that were produced recombinantly were observed after production in prokaryotes and eukaryotes (M. Hynes et al., Neuron 15 (1995) 35-44 and T. Nakamura et al., Biochem. Biophys. Res. Comm. 237 (1997) 465-469).
Hynes et al. compare the activity of hh in the supernatant of transformed human embryonic kidney 293 cells (eukaryotic hh) with hh produced from E. coli and find a four-fold higher activity of hh from the supernatants of the kidney cell line. The reason for this increased activity has been discussed to be a potential additional accessory factor which is only expressed in eukaryotic cells, a post-translational modification, a different N-terminus since the hh isolated from E. coli contains 50% of a hh form which carries two additional N-terminal amino acids (Gly-Ser) or is shortened by 5-6 amino acids, or a higher state of aggregation (e.g. by binding to nickel agarose beads).
Nakamura et al. compare the activity of shh in the supernatant of transformed chicken embryo fibroblasts with an shh fusion protein isolated from E. coli which still has an N-terminal polyhistidine part. The shh in the supernatant of the fibroblasts has a seven-fold higher activity than the purified E. coli protein with regard to stimulation of alkaline phosphatase (AP) in C3H10T 1/2 cells. The increased activity has been postulated to be due to molecules such as for example bone morphogenetic proteins (BMPs) which are only present in the supernatant of eukaryotic cells and cause the stronger induction of AP.
Pepinski et al. (J. Biol. Chem. 273 (1998) 14037-14045) have identified a shh form which is modified with palmitic acid. This shh mutant is 30-fold more potent than the unmodified form in the C3H10T 1/2 assay.
Kinto et al., FEBS Letters, 404 (1997) 319-323 described that fibroblasts which secrete hh induce ectopic bone formation in an i.m. implantation on collagen. Thus hedgehog proteins have an osteoinductive activity. Hedgehog proteins can also stimulate the formation of cartilage cells (Stott et al., 1997).
It is known from Yang et al., Development 124 (1997) 4393-4404 that high local hedgehog concentrations must prevail over a period of at least 16 h at the site of action in the body for a pharmaceutically effective in vivo activity. The carrier system described by Yang et al. i.e. the hedgehog-loaded chromatography medium Affigel CM, the Ni agarose described by Marti et al., in Nature 375 (1995) 322-325 or the Affigel blue used by Lopez-Martinez et al., in Curr.Biol. 5 (1995) 791-796 or the heparin agarose particles that they used are less suitable for a pharmaceutical application since they are immunogenic and can cause inflammatory reactions.